沙库巴曲缬沙坦对糖尿病大鼠心室肌细胞的作用机制研究

Impacts and related mechanisms of sacubitril/valsartan on ventricular myocytes in diabetic rats

目的探讨沙库巴曲缬沙坦对糖尿病大鼠心室肌细胞的保护作用及相关机制。方法选取40只周龄为6~8周的雄性SD大鼠,通过腹腔注射链脲佐菌素建立1型糖尿病大鼠模型,将大鼠分为对照组、糖尿病(DM)组、缬沙坦(DM+Val)组(30 mg·kg^(-1)·d^(-1))及沙库巴曲缬沙坦(DM+Sac/Val)组(60 mg·kg^(-1)·d^(-1))。8周后采用超声心动图检测各组大鼠心功能指标,包括左心室射血分数(LVEF)和左心室短轴缩短率(LVFS),HE染色观察心室肌结构及细胞形态,脱氧核苷酸末端转移酶介导的脱氧尿三磷酸生物素缺口末端标记(TUNEL)染色观察大鼠心室肌细胞凋亡情况,PCR及Western blotting分别检测各组大鼠心室肌组织B淋巴细胞瘤因子相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、C/EBP同源蛋白(CHOP)及葡萄糖调节蛋白78(GRP 78)的mRNA及蛋白表达情况,采用免疫组织化学染色观察GRP 78的蛋白表达情况。采用独立样本t检验、单因素方差分析、Mann‐Whitney U或Kruskal-Wallis H检验进行组间比较。结果TUNEL染色结果显示,与对照组[(0.164±0.048)%]相比,DM组[(2.116±0.305)%]大鼠心室肌细胞凋亡增多;与DM组相比,DM+Val组[(1.121±0.097)%]和DM+Sac/Val组[(0.513±0.031)%]大鼠心室肌细胞凋亡均降低;DM+Sac/Val组较DM+Val组大鼠心室肌细胞凋亡率更低,差异均具有统计学意义(P<0.001)。与对照组相比,DM组大鼠心室肌组织促凋亡蛋白Bax的蛋白及mRNA表达量增加,抑凋亡蛋白Bcl-2的蛋白及mRNA表达量减少,且Bcl-2/Bax降低;与DM组相比,DM+Sac/Val组Bax的蛋白及mRNA表达量降低,蛋白Bcl-2的蛋白及mRNA表达量增加,差异均具有统计学意义(P<0.05)。与对照组相比,DM组大鼠心室肌组织内质网应激相关蛋白GRP 78及CHOP的蛋白及mRNA表达量均增加;与DM组相比,DM+Val组大鼠心室肌组织中CHOP蛋白表达量、GRP 78及CHOP mRNA的表达量均降低,DM+Sac/Val组GRP 78及CHOP的蛋白及mRNA表达量均降低,差异均具有统计学意义(P<0.05)。结论沙库巴曲缬沙坦能够减少糖尿病大鼠心室肌细胞凋亡,改善心脏功能,其作用机制可能与抑制内质网应激相关,且这一效应优于缬沙坦。

Objective To investigate the protective effects and mechanisms of sacubitril/valsartan on ventricular myocytes in diabetic rats.Methods Forty Sprague Dawley(SD)rats aged 6 to 8 weeks were used to establish type 1 diabetic model by intraperitoneal injection of streptozotocin.Rats were divided into control group,diabetes(DM)group,diabetes+valsartan(DM+Val)group(30 mg·kg^(-1)·d^(-1))and diabetes+sacubitril/valsartan(DM+Sac/Val)group(60 mg·kg^(-1)·d^(-1)).After 8 weeks,cardiac function indexes of rats in each group were measured by cardiac ultrasound:left ventricular ejection fractions(LVEF),left ventricular short axis shortening rate(LVFS).Hematoxylin-eosin(HE)staining was used to observe the structure and morphology of ventricular myocytes,and apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuria triphosphase-biotin nick end labeling(TUNEL)staining.Protein and mRNA expression of B-cell lymphoma-2 associated X(Bax),B-cell lymphoma-2(Bcl-2),C/EBP homologous protein(CHOP)and glucose regulatory protein 78(GRP 78)of ventricular myocytes were tested by Western blotting and polymerase chain reaction(PCR),respectively.Immunohistochemical staining was used to observe the expression of GRP 78.Independent-sample t test,one-way analysis of variance(ANOVA),Mann-Whitney U or Kruskal-Wallis H test were used for comparison between groups.Results TUNEL staining showed that apoptosis rate of ventricular myocytes in DM group[(2.116±0.305)%]was increased compared with control group[(0.164±0.048)%].Compared with DM group,apoptosis rate of ventricular myocytes was decreased in DM+Val group[(1.121±0.097)%]and DM+Sac/Val group[(0.513±0.031)%].The apoptosis rate of ventricular myocytes in DM+Sac/Val group was lower than that in DM+Val group(all P<0.001).In comparison with control group,the protein and mRNA expression of pro-apoptotic protein Bax was increased in DM group,while the expression of anti-apoptotic protein Bcl-2 was decreased,and the Bcl-2/Bax ratio was significantly decreased.The protein and mRNA expression of Bax were decreased and the protein and mRNA expression of Bcl-2 were increased in DM+Sac/Val group compared with DM group(all P<0.05).In comparison with control group,the protein and mRNA expressions of endoplasmic reticulum stress-related proteins CHOP and GRP 78 in DM group were significantly increased.Compared with DM group,the protein expression of CHOP and the mRNA expression of CHOP and GRP 78 in DM+Val group were significantly decreased,the protein and mRNA expression of CHOP and GRP 78 in DM+Sac/Val group were significantly decreased(all P<0.05).Conclusions Sacubitril/valsartan reduces the apoptosis of ventricular myocytes and improves cardiac function in diabetic rats,which may be related to the inhibition of endoplasmic reticulum stress.